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Photoinduced biprotonic transfer in 4-methylumbelliferone
Volume 191, number I,2
CHEMICAL PHYSICS LETTERS
27 March 1992
Photoinduced biprotonic transfer in 4-methylumbelliferone Elisabeth Bardez, Patrick Boutin and Bernard Valeur Laboratoire de Chimie G&&ale (CNRS VRA 1103), Conservatoire National des Arts et Mtiers. 292 rue St-Martin, 75003 Paris, France
Received 18 November 199 1; in final form 15 January 1992
Steady-state and time-resolved fluorescence studies of 4-methylumbelliferone in acidic ethanol-water and I-butanol-water mixtures reveal that one water molecule is involved in the rate-limiting step of the phototautomerization process. It is shown that the intermediate species cannot be the cationic form as stated by other authors, but is most likely the anionic form. Moreover, it is concluded that the phototautomer is not zwitterionic but exists only in the neutral keto form. A biprotonic transfer mechanism
1. Introduction Photoinduced tautomerization is the subject of numerous studies [ 1,2] and different pathways of “intramolecular proton transfer” have been substantiated, including true intramolecular proton transfer via vicinal H-bonds, or distal proton transfer by proton relay involving solvent H-bonded bridges. The aim of this paper is to focus on excited-state tautomerization of 7-hydroxy-4-methylcoumarin (4methylumbelliferone: 4-MU). This compound is a fluorescent indicator and a laser dye whose emission range is exceptionally broad (360-590 nm). The prototropic transformations of 4-MU have already
been investigated in aqueous or aqueous-alcoholic solutions, by means of steady-state [ 3-91 or time-resolved fluorometry [ lo- 13 1, but the mechanism of phototautomerization has not been unquestionably cleared up yet. Upon excitation, both the acidity of the phenolic group and the basicity of the carbonyl group of 4-MU are increased relative to the ground state. Then, in the excited state, 4-MU may lead to four possible fluorescent species depending on solvent and pH: the neutral (N*), protonated or cationic (C* ), anionic (A*) forms, and a long-wavelength emitting tautomerit form (T* ), according to general scheme 1. However, only the N, A and C forms exist in the
(neutral ketotautomer)
(zwitterionic form)
hv
Scheme I.
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ground state. The tautomer is an excited-state reaction product, which is formed in such experimental conditions that the absorbing species is the neutral form, i.e. T* arises from N*. The possibility of C* or A* species acting as intermediates in tautomerization has been put forward by some authors, but it cannot be taken for granted. Moreover, the nature of T* itself remains a moot question, because T* may be regarded as a zwitterionic molecule [ 5,131 or a neutral ketotautomer [ 3,4,8,12 1. In this work, attention is directed specifically toward the excited-state tautomerization N*=T* in aqueous-alcoholic solutions, which is reexamined while taking into account the part of the water molecules and of the acidity of the medium in the mechanism. Acidified aqueous-ethanolic or aqueous-nbutanolic solutions turn out to be convenient media for isolating the only emission from N* and T* forms (provided added water concentration is kept sufficiently low). Time-resolved measurements using multifrequency phase fluorometry, and steady-state fluorescence studies are carried out in a complementary manner, allowing the investigation of the kinetics of N* and T* decays, the measurements of the corresponding kinetic constants and the determination of the number of water molecules involved in the tautomerization process.
2. Experimental 4-methylumbelliferone was purchased from Sigma and recrystallized from ethanol. Absolute ethanol and n-butanol were supplied by Merck (pro analysi and spectroscopic grade, respectively). Monodistilled water was further purified by Millipore filtration (conductivity < 10-‘&2-i cm-’ at 25°C). Dye concentrations were typically 5 x 1Oe5 M. In order to get rid of any trace of anionic form in the ground state (4-MU ground state pK= 7.8 [ 5,6,14] ), solutions were acidified by trifluoroacetic acid (from Merck, spectroscopic grade) at a concentration of 0.46 M in all experiments, The emission spectra were recorded using a SLM 8000C spectrofluorometer. They are independent of the excitation wavelength which was selected around the absorption-peak maximum of the neutral form N, i.e. 325 nm.
Time-resolved fluorescence experiments were carried out by means of a multifrequency (0.1-200 MHz) phase-modulation fluorometer described elsewhere [ 15 1. The sample was excited with a HeCd laser beam at 325 nm. The number of frequencies was typically 20-25.
3. Results 3.1. Steady-state fluorescence study in aqueousalcoholic solutions The fluorescence emission spectrum of 4-MU in acidified absolute ethanol or n-butanol without addition of water shows, besides the main band centered around 384 nm and ascribed to the N* form, a much weaker one, around 485 nm, due to T* emission; the acidified alcohols then contain only residual water, the concentration of which is estimated to be 0.09 and 0.02 M in ethanol and butanol, respectively. When the water content in water-alcohol mixtures is increased, the T* band grows up at the expense of N* band, as previously described [ 4,12,13 1. Typical spectral evolution is displayed on fig. 1, in the case of water-ethanol mixture. It is noteworthy that the progressive increase in T* fluorescence for gradual water addition by steps of x 0.3 M as represented here, is weak compared to the very first fluorescence of T*, before any addition of water.
t
N*
-
I 350
4io
470
530
.
590 k l-1
Fig. 1. Corrected fluorescence spectra of 4-MU in aqueous ethanolic solutions with increasing water concentrations from: 0.09 M (residualwater) (1) to4.12M (2).
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Consequently, the tautomerization occurring in the neat alcohols cannot be accounted for by the presence of the little residual water. In fact, by using perchloric acid instead of trifluoroacetic acid to acidify the alcohols, we observed that the extent of tautomerization in the absence of added water depends on the nature of the acid and can be ascribed to the acidity of the medium [ 16 1. However, as seen in fig. 1, further addition of water strongly enhances the excited-state tautomerization. For water concentrations higher than 4.5 M, emission of the anionic form A* at 450 nm begins to superimpose N* and T* fluorescence, indicating that A* ion is then formed in the excited state (no changes occur on the absorption spectrum, the neutral form of 4-MU is only excited species). The slight emission of A* can be detected by making successive differences of the obtained spectra. In the present work, water concentration will thus be kept below 4.5 M in order to observe only the fluorescence of the neutral and tautomeric forms. It is noteworthy that the isoemissive point at 420 nm is not appreciably affected by the appearance of A* emission in addition to the fluorescence of N* and T* species. Observation of the isoemissive point is consequently not a sufficient criterion to make sure of the existence of only two emitting species in the peculiar case of 4MU. As a matter of fact, Abdel-Mottaleb et al. [ 9 ] were misled by the observation of this point in nonacidified water-methanol solutions and missed the identification of the anionic form emission in their spectra. The kinetic scheme of the excited-state reaction which can be proposed to represent the observed phenomena, must take into account that presence of water favours tautomerization in alcohol (scheme 2). Therefore, n water molecules will be considered to be involved in the “intramolecular proton transk N* + n H,O
II k,
=
r
k,
%4
N
Scheme 2.
144
T*
& =
r TT
I
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1992
fer” leading from the N* to T* species. kN and kT denote the reciprocal of the lifetimes of the N* and T* forms, respectively, with the assumption that these parameters remain unchanged for various water contents in a particular water-alcohol mixture. kNT represents the tautomerization rate constant, this reaction being at first assumed to be a one-step reaction. In order to take into account influences of both acidity and water content in the alcohol, kNT can be described by the following empirical expression:
lCN~=kc+Gd-b01”,
(1)
where k,, is the kinetic constant relative to acidity contribution, and k& [ HzO]” the kinetic constant relative to water contribution. The question of the dependence of k,, on water concentration [ HZ0 ] will be further discussed, in light of the results. The kinetic analysis of scheme 2 depends on whether the tautomerization reaction can be considered as reversible or not. Dynamic measurements reveal that the decay of N* fluorescence is monoexponential in the selected experimental conditions (see section 3.2), which means that the back reaction is negligible. The temporal evolution of N* and T* can then be written as [N*(r)
I = [N*loew[ - (h +kNT)tl
(2):
W*lokm [T*(t)1= ,&+,&-kT x{exp(-kTt)-exp[-(kN+kNT)tlj.
(3)
Under continuous illumination, the reciprocal of the stationary concentration in T* form can then be deduced from eq. (3) (4) or
kN ka,+k$TIH,O]”
+’
This equation can be used in the study of the dependence of the T * fluorescence intensity as a function of [ Hz01 . Measurements have been performed in the range [ H,O] ~0-4.5 M as aforementioned, and at wavelengths longer than 500 nm in order to minimize any residual contribution of N* emission
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in the measured signal. The obtained results are similar in both water-alcohol mixtures. In the range of water concentration l-4.5 M, the plot of l/ [T*] as a function of l/ [H,O] displays a clear linear dependence (fig. 2) which indicates that (i) the value of n is 1 and (ii) the term kc can be disregarded with respect to k& [ HZ01 . Hence, in this range of water concentration eq. (5 ) can be simplified according to
1/ tT*l=k-r Consequently, extrapolation of the plot of fig. 2 which gives the ratio intercept/slope, leads to k&,/k,= 0.34-0.35 M-i in both water-alcohol mixtures. These two kinetic parameters will be independently determined by means of time-resolved data. As regards k,,, its value when [ Hz0 ] -+0 cannot be deduced from these experiments, but it will be inferred from the dynamic studies. The study of T* fluorescence intensity has thus allowed us to point out that tautomerization which occurs in neat acidic ethanol or butanol, is enhanced by addition of water. When [ Hz01 > 1 M (i.e. 2% v/ v), the contribution of water becomes kinetically predominant. The outstanding value of one water molecule involved in the tautomerization of one dye molecule is a salient feature, borne out by dynamic measurements, and discussed hereafter.
27 March I992
3.2. Time-resolved fluorescence study in aqueowalcoholic solutions In the time-resolved studies by multifrequency phase-modulation fluorometry, the fluorescence of the neutral and tautomeric forms of 4-MU were selected at 366 nm with an interferential filter, and at 1> 530 nm with a cutoff filter, respectively. The phase and modulation data relevant to the neutral form show that the decay of N* is unambiguously a single exponential in the range of water concentration l-4.5 M, which means that, under these conditions, the back reaction cannot occur during the lifetime of the excited state, as previously reported [ 12,13 1. It is observed that the rate constant of this decay (k,,, = kN + kNT) gradually increases upon addition of water. Assuming that kN is not significantly affected by addition of water (vide supra), this observation corroborates that the presence of water enhances kNr, i.e. the tautomerization process. According to eqs. ( 1) and (2 ), the decay rate constant is given by k,=k,+&,+kO,,[H,O]“.
(7)
The linearity of the plots of k,,, versus [H,O] (fig. 3 ) shows that n = 1, consistently with the steady-state study. Besides a linear dependence of the decay time of the neutral form N* on water concentration in aqueous-ethanolic solutions has already been reported [ 12 1. This result provides compelling evi-
lo-’ k m 1.51
l/F(T.,t
-0.5
6
EtOH / Eau
IS-‘1
015 IH,Ol
Fig. 2. Reciprocal of T* fluorescence intensity versus reciprocal of water concentration. Measurements performed at: 500 nm (0),52Onm(0),540nm(+).
Ml
Fig. 3. Variations in decay rate constant of the N* form (&) versus water concentration in: aqueous-ethanolic mixtures ( 0 ) and aqueous-butanolic mixtures ( 0 ).
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Table 1 Lifetimes and rate constants Ethanol
I-butanol
T,(ns) TT(ns)
2.17 5.22
2.41 5.40
kN(s-‘) kT(s-‘) k&(M-Is-‘) kx(s-‘)
4.5x 1.9x 1.5x 2.8x
lo8 lo8 lo8 lo*
4.0x lo8 1.8sx lo8 1.4x10* 2.0x IO8
dence that one water molecule is involved in the ratelimiting process (see discussion). From the linear plots, the values of the intercept ( kN+ k,,) and the slope (k&r ) can be deduced. Combining these results with those from the steady-state study, it is possible to calculate all the rate constants and the lifetime of the neutral form (table 1). Under the assumption that the excited tautomer T* can be formed only from the excited neutral form N* by a one-step reaction, the time evolution of T* is a difference of two exponentials. In phase-modulation fluorometry, it has long been recognized [ 171 that, when the initially excited fluorescent centers A* form new emitting centers B* during the lifetime of their excited states, the phase difference &. - $A*and the modulation ratio M&M.,,. characterize the intrinsic decay of centers B*, i.e. the decay of B* molecules if they were excited directly. Therefore, after measuring the phase shift tiT’ and the modulation MT* of the tautomer at the same frequencies as those used for the neutral form, & - & and MT./MN. are calculated at each modulation frequency and the resulting data are processed in the usual way. The intrinsic decay of T* is found to be monoexponential and, since there is no back reaction, the rate constant of this decay represents the reciprocal of the lifetime rr of the tautomer. The lifetime values obtained in this way are independent of the amount of water, which validates the above assumption. The mean values of T, are 5.22 +0.02 ns in ethanol and 5.40 + 0.02 ns in butanol (table 1).
4. Discussion The value found for the lifetime of T* in ethanolwater mixtures is consistent with the results of Balter 146
27 March 1992
and Rolinski [ 13 ] though perchloric acid was used in their study instead of trifluoroacetic acid. On the contrary, the values of the neutral form lifetime obtained in the present study are larger than those previously reported in this mixed solvent ( rN= 1.35 ns [ 121, 1.6 ns [ 131). This discrepancy is due to the fact that our expression for tautomerization rate constant takes into account the rate constant k,, of the reaction in neat alcohols, the extent of which depends on the acidity of the medium. Even in lack of water, tautomerization competes with N* emission. This phenomenon becomes negligible with respect to water contribution for [H,O] > 1 M, which can explain that the acid concentration could be considered as having little effect on the phenomena reported by Bauer and Kowalczyk [ 12 1. The point is now to examine our results in further detail in order to get information on the nature of the tautomer and the mechanism of its formation. Schematically, two limiting structures of the tautomerit form are to be considered, either zwitterionic or neutral keto-form. Let us examine successively these two possibilities. The zwitterionic form results from protonation and deprotonation processes occurring in two steps. Depending on the sequence of the two processes, either the anionic A* or cationic C* forms are intermediates in the two-step excited reaction according to scheme 1. Balter and Rolinski, presuming a zwitterionic structure for T*, came to the conclusion that C* was the intermediate species, whose dissociation rate is very high compared to its formation rate [ 13 ] ; this means that the measured rate constant for tautomerization represents the protonation rate of the excited neutral form according to the stationary state approximation. The corresponding rate constant may then be written as kd [ H+ ] in accordance with a diffusional protonation process. This assumption is not consistent with the empirical form kNT= k,, + k&[H,O] (or kNT=k&[H20] when [HZ01 > 1 M) that the present study has substantiated. In particular, the involvement of one water molecule in the reaction is deprived of any physical meaning if the observed phenomenon is the protonation of the N* form. The other way of formation of the zwitterion occurs via the anionic A* intermediate, and consists of the sequence of deprotonation and protonation. It is
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noteworthy that the acidity of the phenolic part of 4MU in the excited molecule is enhanced in such a manner (excited-state pP~O.45 [ 61) that deprotonation is not impaired by the acidity of the medium (0.46 M in CF,COOH). No observable A* emission implies again a stationary state approximation for A* species, and then the measured rate constant corresponds to the first step, i.e. proton extraction by a water molecule according to N*+H20+A*+H,0+. It can be written as kd[ H20] under the assumption of a diffusional process of the water molecule. One water molecule is then concerned in the kinetic law, in accordance with our results. This first step is ratelimiting in the overall process, so that the temporal evolution of the excited species is the same as in a one-step reaction, as we have observed. Moreover, the occurrence of the anionic form as an intermediate is consistent with the appearance of A* emission when the amount of water exceeds 4.5 M. Then, one would expect the water concentration to be high enough so that deprotonation becomes faster than protonation, the intermediate A* being no more in a stationary state. However, even if the hypothesis of a two-step tautomerization involving A* as an intermediate species is now in agreement with the experimental results, it does not necessarily imply that the tautomeric form is a zwitterion. In the previous studies, the proposed mechanisms leading to a neutral keto-tautomer favour either a direct conversion to T* by means of a concerted proton transfer from the hydroxyl site to the carbonyl site [ 8 1, or “dissociative” two-step ways via A* or C* species [ 3,121. The possibility of a proton relay with the aid of hydrogen-bonded solvent molecules can be discarded in the present experimental conditions because a relay implies more than one molecule of water. For example, Itoh et al. [ 18 ] demonstrated that two molecules of methanol are responsible for tautomerization of 7-hydroxyquinoline in hexane. On the contrary, a two-step reaction via A* species, in agreement with our results, can produce a neutral tautomer if the anionic form is stabilized by several structures and is protonated under the enolate form according to scheme 3. Such a model accounts more likely for a fast second-step protonation because protonation then occurs on the
27 March 1992
FAST
o&o_ oJ&oHG Scheme 3.
anionic enolate site rather than on the carbonyl site, as would be the case for a zwitterionic tautomer. Moreover, further evidence for the existence of the tautomer under the neutral keto-form is provided by experiments carried out in water-alcohol mixtures with various alcohols of different polarity: methanol, ethanol, n-butanol, n-pentanol, n-decanol. The tautomer emission band does not undergo a red-shift when increasing the polarity of the solvent, as would be expected for a zwitterionic form because of solvent relaxation arising from a large increase in the dipole moment with respect to the ground-state neutral form. In conclusion, the phototautomerization of 4-MU is most likely to be a biprotonic transfer in which the first step is rate limited by the diffusion of one water molecule towards the phenolic group, leading to deprotonation of the neutral form into the intermediate anionic A* form. Then, rapid electronic reorganization of A* occurs, and a fast protonation of the enolate limiting structure yields the neutral keto-tautomer of the initially excited neutral form of the dye.
References [ I ] M. Rasha, J. Chem. Sot. Faraday Trans. II 82 ( 1986) 2379. [ 21 V. Balzani and F. Scandola, Supramolecular photochemistry (Ellis Horwood, Chichester, 199 1) p. 342. [ 31 C.V. Shank, A. Dienes, A.M. Trozzolo and J.A. Myer, Appl. Phys. Letters 16 ( 1970) 405; A. Dienes, C.V. Shank and A.M. Trozzolo, Appl. Phys. Letters 17 (1970) 189; A.M. Trozzolo, A. Dienes and C.V. Shank, J. Am. Chem. Sot. 96 (1974) 4699. [4] M. Nakashima, J.A. Sousa and R.C. Clapp, Nature Phys. Sci. 235 (1972) 16.
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[ 5) G.J. Yakatan, R.J. Juneau and S.G. Schulman, Anal. Chem. 44 (1972) 1044; S.G. Schulman and L.S. Rosenberg, J. Phys. Chem. 83 (1979) 447. [6] V. Mikes, Collection Czech. Chem. Commun. 44 ( 1979) 508. [ 71 O.A. Ponomarev, E.R. Vasina, S.N. Yarmolenko and V.G. Mitina, Zh. Obshch. Khim. 55 (1985) 158. [8] T. Moriya, Bull. Chem. Sot. Japan 61 (1988) 1873. [ 91 M.S.A. Abdel-Mottaleb, B.A. El-Sayed, M.M. Abo-Aly and M.Y. El-Kady, J. Photochem. Photobiol. A 46 (1989) 379. [ lo] P.E. Zinsli, H.P. Tschanz, 0. Jenni and Th. Binkert, Isv. Akad. Nauk SSSR. Ser. Fiz. 37 (1973) 391; P.E. Zinsli, J. Photochem. 3 (1974/1975) 55.
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[ 1 I ] T. Kobayashi, J. Phys. Chem. 82 ( 1978) 2277.
[ 121 R.K. Bauer and A. Kowalczyk, Z. Naturforsch. 35a (1980) 946.
[ 131 A. Balter and 0. Rolinski, Z. Naturforsch. 39a ( 1984) 1035. [ 141 J.C. Lechevin, Bull. Trav. Sot. Pharm. Lyon 26 (1982) 48. [ 151 J. Pouget, J. Mugnier and B. Valeur, J. Phys. E 22 (1989) 855.
[ 161 E. Bardez, P. Boutin and B. Valeur, unpublished results. [ 171 J.R. Iakowicz and A. Balter, Biophys. Chem. 16 (1982) 99, and references therein. [ 181 M. Itoh, T. Adachi and K. Tokumura, J. Am. Chem. Sot. 106 (1984) 850.
CHEMICAL PHYSICS LETTERS
27 March 1992
Photoinduced biprotonic transfer in 4-methylumbelliferone Elisabeth Bardez, Patrick Boutin and Bernard Valeur Laboratoire de Chimie G&&ale (CNRS VRA 1103), Conservatoire National des Arts et Mtiers. 292 rue St-Martin, 75003 Paris, France
Received 18 November 199 1; in final form 15 January 1992
Steady-state and time-resolved fluorescence studies of 4-methylumbelliferone in acidic ethanol-water and I-butanol-water mixtures reveal that one water molecule is involved in the rate-limiting step of the phototautomerization process. It is shown that the intermediate species cannot be the cationic form as stated by other authors, but is most likely the anionic form. Moreover, it is concluded that the phototautomer is not zwitterionic but exists only in the neutral keto form. A biprotonic transfer mechanism
1. Introduction Photoinduced tautomerization is the subject of numerous studies [ 1,2] and different pathways of “intramolecular proton transfer” have been substantiated, including true intramolecular proton transfer via vicinal H-bonds, or distal proton transfer by proton relay involving solvent H-bonded bridges. The aim of this paper is to focus on excited-state tautomerization of 7-hydroxy-4-methylcoumarin (4methylumbelliferone: 4-MU). This compound is a fluorescent indicator and a laser dye whose emission range is exceptionally broad (360-590 nm). The prototropic transformations of 4-MU have already
been investigated in aqueous or aqueous-alcoholic solutions, by means of steady-state [ 3-91 or time-resolved fluorometry [ lo- 13 1, but the mechanism of phototautomerization has not been unquestionably cleared up yet. Upon excitation, both the acidity of the phenolic group and the basicity of the carbonyl group of 4-MU are increased relative to the ground state. Then, in the excited state, 4-MU may lead to four possible fluorescent species depending on solvent and pH: the neutral (N*), protonated or cationic (C* ), anionic (A*) forms, and a long-wavelength emitting tautomerit form (T* ), according to general scheme 1. However, only the N, A and C forms exist in the
(neutral ketotautomer)
(zwitterionic form)
hv
Scheme I.
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ground state. The tautomer is an excited-state reaction product, which is formed in such experimental conditions that the absorbing species is the neutral form, i.e. T* arises from N*. The possibility of C* or A* species acting as intermediates in tautomerization has been put forward by some authors, but it cannot be taken for granted. Moreover, the nature of T* itself remains a moot question, because T* may be regarded as a zwitterionic molecule [ 5,131 or a neutral ketotautomer [ 3,4,8,12 1. In this work, attention is directed specifically toward the excited-state tautomerization N*=T* in aqueous-alcoholic solutions, which is reexamined while taking into account the part of the water molecules and of the acidity of the medium in the mechanism. Acidified aqueous-ethanolic or aqueous-nbutanolic solutions turn out to be convenient media for isolating the only emission from N* and T* forms (provided added water concentration is kept sufficiently low). Time-resolved measurements using multifrequency phase fluorometry, and steady-state fluorescence studies are carried out in a complementary manner, allowing the investigation of the kinetics of N* and T* decays, the measurements of the corresponding kinetic constants and the determination of the number of water molecules involved in the tautomerization process.
2. Experimental 4-methylumbelliferone was purchased from Sigma and recrystallized from ethanol. Absolute ethanol and n-butanol were supplied by Merck (pro analysi and spectroscopic grade, respectively). Monodistilled water was further purified by Millipore filtration (conductivity < 10-‘&2-i cm-’ at 25°C). Dye concentrations were typically 5 x 1Oe5 M. In order to get rid of any trace of anionic form in the ground state (4-MU ground state pK= 7.8 [ 5,6,14] ), solutions were acidified by trifluoroacetic acid (from Merck, spectroscopic grade) at a concentration of 0.46 M in all experiments, The emission spectra were recorded using a SLM 8000C spectrofluorometer. They are independent of the excitation wavelength which was selected around the absorption-peak maximum of the neutral form N, i.e. 325 nm.
Time-resolved fluorescence experiments were carried out by means of a multifrequency (0.1-200 MHz) phase-modulation fluorometer described elsewhere [ 15 1. The sample was excited with a HeCd laser beam at 325 nm. The number of frequencies was typically 20-25.
3. Results 3.1. Steady-state fluorescence study in aqueousalcoholic solutions The fluorescence emission spectrum of 4-MU in acidified absolute ethanol or n-butanol without addition of water shows, besides the main band centered around 384 nm and ascribed to the N* form, a much weaker one, around 485 nm, due to T* emission; the acidified alcohols then contain only residual water, the concentration of which is estimated to be 0.09 and 0.02 M in ethanol and butanol, respectively. When the water content in water-alcohol mixtures is increased, the T* band grows up at the expense of N* band, as previously described [ 4,12,13 1. Typical spectral evolution is displayed on fig. 1, in the case of water-ethanol mixture. It is noteworthy that the progressive increase in T* fluorescence for gradual water addition by steps of x 0.3 M as represented here, is weak compared to the very first fluorescence of T*, before any addition of water.
t
N*
-
I 350
4io
470
530
.
590 k l-1
Fig. 1. Corrected fluorescence spectra of 4-MU in aqueous ethanolic solutions with increasing water concentrations from: 0.09 M (residualwater) (1) to4.12M (2).
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Consequently, the tautomerization occurring in the neat alcohols cannot be accounted for by the presence of the little residual water. In fact, by using perchloric acid instead of trifluoroacetic acid to acidify the alcohols, we observed that the extent of tautomerization in the absence of added water depends on the nature of the acid and can be ascribed to the acidity of the medium [ 16 1. However, as seen in fig. 1, further addition of water strongly enhances the excited-state tautomerization. For water concentrations higher than 4.5 M, emission of the anionic form A* at 450 nm begins to superimpose N* and T* fluorescence, indicating that A* ion is then formed in the excited state (no changes occur on the absorption spectrum, the neutral form of 4-MU is only excited species). The slight emission of A* can be detected by making successive differences of the obtained spectra. In the present work, water concentration will thus be kept below 4.5 M in order to observe only the fluorescence of the neutral and tautomeric forms. It is noteworthy that the isoemissive point at 420 nm is not appreciably affected by the appearance of A* emission in addition to the fluorescence of N* and T* species. Observation of the isoemissive point is consequently not a sufficient criterion to make sure of the existence of only two emitting species in the peculiar case of 4MU. As a matter of fact, Abdel-Mottaleb et al. [ 9 ] were misled by the observation of this point in nonacidified water-methanol solutions and missed the identification of the anionic form emission in their spectra. The kinetic scheme of the excited-state reaction which can be proposed to represent the observed phenomena, must take into account that presence of water favours tautomerization in alcohol (scheme 2). Therefore, n water molecules will be considered to be involved in the “intramolecular proton transk N* + n H,O
II k,
=
r
k,
%4
N
Scheme 2.
144
T*
& =
r TT
I
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1992
fer” leading from the N* to T* species. kN and kT denote the reciprocal of the lifetimes of the N* and T* forms, respectively, with the assumption that these parameters remain unchanged for various water contents in a particular water-alcohol mixture. kNT represents the tautomerization rate constant, this reaction being at first assumed to be a one-step reaction. In order to take into account influences of both acidity and water content in the alcohol, kNT can be described by the following empirical expression:
lCN~=kc+Gd-b01”,
(1)
where k,, is the kinetic constant relative to acidity contribution, and k& [ HzO]” the kinetic constant relative to water contribution. The question of the dependence of k,, on water concentration [ HZ0 ] will be further discussed, in light of the results. The kinetic analysis of scheme 2 depends on whether the tautomerization reaction can be considered as reversible or not. Dynamic measurements reveal that the decay of N* fluorescence is monoexponential in the selected experimental conditions (see section 3.2), which means that the back reaction is negligible. The temporal evolution of N* and T* can then be written as [N*(r)
I = [N*loew[ - (h +kNT)tl
(2):
W*lokm [T*(t)1= ,&+,&-kT x{exp(-kTt)-exp[-(kN+kNT)tlj.
(3)
Under continuous illumination, the reciprocal of the stationary concentration in T* form can then be deduced from eq. (3) (4) or
kN ka,+k$TIH,O]”
+’
This equation can be used in the study of the dependence of the T * fluorescence intensity as a function of [ Hz01 . Measurements have been performed in the range [ H,O] ~0-4.5 M as aforementioned, and at wavelengths longer than 500 nm in order to minimize any residual contribution of N* emission
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in the measured signal. The obtained results are similar in both water-alcohol mixtures. In the range of water concentration l-4.5 M, the plot of l/ [T*] as a function of l/ [H,O] displays a clear linear dependence (fig. 2) which indicates that (i) the value of n is 1 and (ii) the term kc can be disregarded with respect to k& [ HZ01 . Hence, in this range of water concentration eq. (5 ) can be simplified according to
1/ tT*l=k-r Consequently, extrapolation of the plot of fig. 2 which gives the ratio intercept/slope, leads to k&,/k,= 0.34-0.35 M-i in both water-alcohol mixtures. These two kinetic parameters will be independently determined by means of time-resolved data. As regards k,,, its value when [ Hz0 ] -+0 cannot be deduced from these experiments, but it will be inferred from the dynamic studies. The study of T* fluorescence intensity has thus allowed us to point out that tautomerization which occurs in neat acidic ethanol or butanol, is enhanced by addition of water. When [ Hz01 > 1 M (i.e. 2% v/ v), the contribution of water becomes kinetically predominant. The outstanding value of one water molecule involved in the tautomerization of one dye molecule is a salient feature, borne out by dynamic measurements, and discussed hereafter.
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3.2. Time-resolved fluorescence study in aqueowalcoholic solutions In the time-resolved studies by multifrequency phase-modulation fluorometry, the fluorescence of the neutral and tautomeric forms of 4-MU were selected at 366 nm with an interferential filter, and at 1> 530 nm with a cutoff filter, respectively. The phase and modulation data relevant to the neutral form show that the decay of N* is unambiguously a single exponential in the range of water concentration l-4.5 M, which means that, under these conditions, the back reaction cannot occur during the lifetime of the excited state, as previously reported [ 12,13 1. It is observed that the rate constant of this decay (k,,, = kN + kNT) gradually increases upon addition of water. Assuming that kN is not significantly affected by addition of water (vide supra), this observation corroborates that the presence of water enhances kNr, i.e. the tautomerization process. According to eqs. ( 1) and (2 ), the decay rate constant is given by k,=k,+&,+kO,,[H,O]“.
(7)
The linearity of the plots of k,,, versus [H,O] (fig. 3 ) shows that n = 1, consistently with the steady-state study. Besides a linear dependence of the decay time of the neutral form N* on water concentration in aqueous-ethanolic solutions has already been reported [ 12 1. This result provides compelling evi-
lo-’ k m 1.51
l/F(T.,t
-0.5
6
EtOH / Eau
IS-‘1
015 IH,Ol
Fig. 2. Reciprocal of T* fluorescence intensity versus reciprocal of water concentration. Measurements performed at: 500 nm (0),52Onm(0),540nm(+).
Ml
Fig. 3. Variations in decay rate constant of the N* form (&) versus water concentration in: aqueous-ethanolic mixtures ( 0 ) and aqueous-butanolic mixtures ( 0 ).
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Table 1 Lifetimes and rate constants Ethanol
I-butanol
T,(ns) TT(ns)
2.17 5.22
2.41 5.40
kN(s-‘) kT(s-‘) k&(M-Is-‘) kx(s-‘)
4.5x 1.9x 1.5x 2.8x
lo8 lo8 lo8 lo*
4.0x lo8 1.8sx lo8 1.4x10* 2.0x IO8
dence that one water molecule is involved in the ratelimiting process (see discussion). From the linear plots, the values of the intercept ( kN+ k,,) and the slope (k&r ) can be deduced. Combining these results with those from the steady-state study, it is possible to calculate all the rate constants and the lifetime of the neutral form (table 1). Under the assumption that the excited tautomer T* can be formed only from the excited neutral form N* by a one-step reaction, the time evolution of T* is a difference of two exponentials. In phase-modulation fluorometry, it has long been recognized [ 171 that, when the initially excited fluorescent centers A* form new emitting centers B* during the lifetime of their excited states, the phase difference &. - $A*and the modulation ratio M&M.,,. characterize the intrinsic decay of centers B*, i.e. the decay of B* molecules if they were excited directly. Therefore, after measuring the phase shift tiT’ and the modulation MT* of the tautomer at the same frequencies as those used for the neutral form, & - & and MT./MN. are calculated at each modulation frequency and the resulting data are processed in the usual way. The intrinsic decay of T* is found to be monoexponential and, since there is no back reaction, the rate constant of this decay represents the reciprocal of the lifetime rr of the tautomer. The lifetime values obtained in this way are independent of the amount of water, which validates the above assumption. The mean values of T, are 5.22 +0.02 ns in ethanol and 5.40 + 0.02 ns in butanol (table 1).
4. Discussion The value found for the lifetime of T* in ethanolwater mixtures is consistent with the results of Balter 146
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and Rolinski [ 13 ] though perchloric acid was used in their study instead of trifluoroacetic acid. On the contrary, the values of the neutral form lifetime obtained in the present study are larger than those previously reported in this mixed solvent ( rN= 1.35 ns [ 121, 1.6 ns [ 131). This discrepancy is due to the fact that our expression for tautomerization rate constant takes into account the rate constant k,, of the reaction in neat alcohols, the extent of which depends on the acidity of the medium. Even in lack of water, tautomerization competes with N* emission. This phenomenon becomes negligible with respect to water contribution for [H,O] > 1 M, which can explain that the acid concentration could be considered as having little effect on the phenomena reported by Bauer and Kowalczyk [ 12 1. The point is now to examine our results in further detail in order to get information on the nature of the tautomer and the mechanism of its formation. Schematically, two limiting structures of the tautomerit form are to be considered, either zwitterionic or neutral keto-form. Let us examine successively these two possibilities. The zwitterionic form results from protonation and deprotonation processes occurring in two steps. Depending on the sequence of the two processes, either the anionic A* or cationic C* forms are intermediates in the two-step excited reaction according to scheme 1. Balter and Rolinski, presuming a zwitterionic structure for T*, came to the conclusion that C* was the intermediate species, whose dissociation rate is very high compared to its formation rate [ 13 ] ; this means that the measured rate constant for tautomerization represents the protonation rate of the excited neutral form according to the stationary state approximation. The corresponding rate constant may then be written as kd [ H+ ] in accordance with a diffusional protonation process. This assumption is not consistent with the empirical form kNT= k,, + k&[H,O] (or kNT=k&[H20] when [HZ01 > 1 M) that the present study has substantiated. In particular, the involvement of one water molecule in the reaction is deprived of any physical meaning if the observed phenomenon is the protonation of the N* form. The other way of formation of the zwitterion occurs via the anionic A* intermediate, and consists of the sequence of deprotonation and protonation. It is
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noteworthy that the acidity of the phenolic part of 4MU in the excited molecule is enhanced in such a manner (excited-state pP~O.45 [ 61) that deprotonation is not impaired by the acidity of the medium (0.46 M in CF,COOH). No observable A* emission implies again a stationary state approximation for A* species, and then the measured rate constant corresponds to the first step, i.e. proton extraction by a water molecule according to N*+H20+A*+H,0+. It can be written as kd[ H20] under the assumption of a diffusional process of the water molecule. One water molecule is then concerned in the kinetic law, in accordance with our results. This first step is ratelimiting in the overall process, so that the temporal evolution of the excited species is the same as in a one-step reaction, as we have observed. Moreover, the occurrence of the anionic form as an intermediate is consistent with the appearance of A* emission when the amount of water exceeds 4.5 M. Then, one would expect the water concentration to be high enough so that deprotonation becomes faster than protonation, the intermediate A* being no more in a stationary state. However, even if the hypothesis of a two-step tautomerization involving A* as an intermediate species is now in agreement with the experimental results, it does not necessarily imply that the tautomeric form is a zwitterion. In the previous studies, the proposed mechanisms leading to a neutral keto-tautomer favour either a direct conversion to T* by means of a concerted proton transfer from the hydroxyl site to the carbonyl site [ 8 1, or “dissociative” two-step ways via A* or C* species [ 3,121. The possibility of a proton relay with the aid of hydrogen-bonded solvent molecules can be discarded in the present experimental conditions because a relay implies more than one molecule of water. For example, Itoh et al. [ 18 ] demonstrated that two molecules of methanol are responsible for tautomerization of 7-hydroxyquinoline in hexane. On the contrary, a two-step reaction via A* species, in agreement with our results, can produce a neutral tautomer if the anionic form is stabilized by several structures and is protonated under the enolate form according to scheme 3. Such a model accounts more likely for a fast second-step protonation because protonation then occurs on the
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FAST
o&o_ oJ&oHG Scheme 3.
anionic enolate site rather than on the carbonyl site, as would be the case for a zwitterionic tautomer. Moreover, further evidence for the existence of the tautomer under the neutral keto-form is provided by experiments carried out in water-alcohol mixtures with various alcohols of different polarity: methanol, ethanol, n-butanol, n-pentanol, n-decanol. The tautomer emission band does not undergo a red-shift when increasing the polarity of the solvent, as would be expected for a zwitterionic form because of solvent relaxation arising from a large increase in the dipole moment with respect to the ground-state neutral form. In conclusion, the phototautomerization of 4-MU is most likely to be a biprotonic transfer in which the first step is rate limited by the diffusion of one water molecule towards the phenolic group, leading to deprotonation of the neutral form into the intermediate anionic A* form. Then, rapid electronic reorganization of A* occurs, and a fast protonation of the enolate limiting structure yields the neutral keto-tautomer of the initially excited neutral form of the dye.
References [ I ] M. Rasha, J. Chem. Sot. Faraday Trans. II 82 ( 1986) 2379. [ 21 V. Balzani and F. Scandola, Supramolecular photochemistry (Ellis Horwood, Chichester, 199 1) p. 342. [ 31 C.V. Shank, A. Dienes, A.M. Trozzolo and J.A. Myer, Appl. Phys. Letters 16 ( 1970) 405; A. Dienes, C.V. Shank and A.M. Trozzolo, Appl. Phys. Letters 17 (1970) 189; A.M. Trozzolo, A. Dienes and C.V. Shank, J. Am. Chem. Sot. 96 (1974) 4699. [4] M. Nakashima, J.A. Sousa and R.C. Clapp, Nature Phys. Sci. 235 (1972) 16.
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[ 5) G.J. Yakatan, R.J. Juneau and S.G. Schulman, Anal. Chem. 44 (1972) 1044; S.G. Schulman and L.S. Rosenberg, J. Phys. Chem. 83 (1979) 447. [6] V. Mikes, Collection Czech. Chem. Commun. 44 ( 1979) 508. [ 71 O.A. Ponomarev, E.R. Vasina, S.N. Yarmolenko and V.G. Mitina, Zh. Obshch. Khim. 55 (1985) 158. [8] T. Moriya, Bull. Chem. Sot. Japan 61 (1988) 1873. [ 91 M.S.A. Abdel-Mottaleb, B.A. El-Sayed, M.M. Abo-Aly and M.Y. El-Kady, J. Photochem. Photobiol. A 46 (1989) 379. [ lo] P.E. Zinsli, H.P. Tschanz, 0. Jenni and Th. Binkert, Isv. Akad. Nauk SSSR. Ser. Fiz. 37 (1973) 391; P.E. Zinsli, J. Photochem. 3 (1974/1975) 55.
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[ 1 I ] T. Kobayashi, J. Phys. Chem. 82 ( 1978) 2277.
[ 121 R.K. Bauer and A. Kowalczyk, Z. Naturforsch. 35a (1980) 946.
[ 131 A. Balter and 0. Rolinski, Z. Naturforsch. 39a ( 1984) 1035. [ 141 J.C. Lechevin, Bull. Trav. Sot. Pharm. Lyon 26 (1982) 48. [ 151 J. Pouget, J. Mugnier and B. Valeur, J. Phys. E 22 (1989) 855.
[ 161 E. Bardez, P. Boutin and B. Valeur, unpublished results. [ 171 J.R. Iakowicz and A. Balter, Biophys. Chem. 16 (1982) 99, and references therein. [ 181 M. Itoh, T. Adachi and K. Tokumura, J. Am. Chem. Sot. 106 (1984) 850.